Assaying ovarian cyst fluid

ABSTRACT

A diagnostic test for ovarian cysts is based on the detection of mutations characteristic of the most common neoplasms giving rise to these lesions. With this test, tumor-specific mutations were detected in the cyst fluids of 19 of 24 (79%) borderline tumors and 28 of 31 (90%) malignant ovarian cancers. In contrast, we detected no mutations in the cyst fluids from 10 non-neoplastic cysts and 12 benign tumors. When categorized by the need for exploratory surgery (i.e., presence of a borderline tumor or malignant cancer), the sensitivity of this test was 85% and the specificity was 100%. These tests could inform the diagnosis of ovarian cysts and improve the clinical management of the large number of women with these lesions.

This application is a Continuation of U.S. application Ser. No.15/749,887, filed Feb. 2, 2018, which is a National Stage applicationunder 35 U.S.C. § 371 of International Application No.PCT/US2016/046453, having an International Filing Date of Aug. 11, 2016,which claims the benefit of U.S. Provisional Application No. 62/203,573,filed Aug. 11, 2015, each of which is incorporated herein by referencein its entirety.

This invention was made with government support under CA 043460, 057345,and 062924 awarded by National Institutes of Health. The government hascertain rights in the invention.

TECHNICAL FIELD OF THE INVENTION

This invention is related to the area of DNA analysis. In particular, itrelates to analysis of genes in clinical samples.

BACKGROUND OF THE INVENTION

Ovarian cancer is the most lethal gynecologic malignancy, with 21,980estimated new cases and 14,270 estimated deaths in the United States in2014. Approximately 1.3% of women will be diagnosed with ovarian cancerduring their lifetime (1). These cancers commonly present as an adnexalmass with cystic components, but are not associated with specificsymptoms. As a result, two-thirds of ovarian cancers are diagnosed atlate stage (Stage III and IV), when the 5-year survival is less than 30%(1).

Complicating the diagnosis of ovarian cancer is the fact that ovariancysts are common in women of all ages, with a prevalence of 35% and 17%in pre- and post-menopausal women, respectively (2). These cysts arefrequently benign and found incidentally on routine imaging (2). Thoughmalignancy is an unusual cause of the cysts, 30% of the cysts exhibitradiographic features suspicious for malignancy, such as solid areas ormass (2). In addition to the anxiety that such findings provoke, manywomen undergo unnecessary surgery for cysts that are not malignant andmay not be responsible for the symptoms they have. For example, only 5%of 570 women in a large ovarian cancer screening randomized trial whounderwent surgical evaluation actually had a malignancy (3). Compoundingthis issue is the fact that surgery for ovarian cysts requires generalanesthesia and is associated with significant morbidity, causing seriouscomplications in 15% of women. These complications include damage tonerves and ureters, bleeding, infection, perforation of adjacentviscera, as well as hormonal and fertility loss (in the case ofbilateral oophorectomy) (4). Even minimal procedures such as ovariancystectomy can affect fertility in premenopausal women by decreasingfollicular response and oocyte number (5, 6). If a preoperative testcould be performed that indicated whether the cystic lesion was benignor malignant, unnecessary surgery and its associated complications couldbe avoided in a large number of patients, particularly women ofreproductive age who wish to preserve their fertility, as well as womenwhose medical comorbidities or functional status makes anesthesia andsurgery hazardous.

Ovarian cysts and tumors are classified as non-neoplastic, benign,borderline, or malignant based on microscopic examination after surgicalremoval (FIG. 1). Non-neoplastic cysts are by far the most common classof ovarian cyst. They are frequently functional in pre-menopausal women,arising when an egg is not released properly from either the follicle orcorpus luteum and usually resolve spontaneously within several months(7). Benign cystic tumors, such as cystadenomas and cystadenofibromas,rarely progress to malignancy (8, 9). No genetic alterations have yetbeen identified in either non-neoplastic cysts or in benign cystictumors (9). Neither of these cyst types requires surgery unless they aresymptomatic or have undergone torsion (8). These cysts can be easilysampled with ultrasound-guided fine-needle aspiration in an outpatientsetting without the need for anesthesia (10).

At the other end of the spectrum are epithelial ovarian cancers, whichare potentially lethal and unequivocally require surgery. A dualisticmodel has been proposed to classify these neoplasms (11). Type I tumorsare composed of low-grade serous, low-grade endometrioid, clear cell,and mucinous carcinomas. They are clinically indolent, frequentlydiagnosed at early stage (Stage I or II), and develop fromwell-established precursor lesions (“borderline” or “atypicalproliferative” tumors, as described below) (12). Type I cancers commonlyexhibit mutations in KRAS, BRAF, CTNNB1, PIK3CA, PTEN, ARID1A, orPPP2R1A (11). In contrast, type II tumors are generally high-gradeserous carcinomas. They are highly aggressive, most often diagnosed inlate stage (Stage III or IV), and have suggested origins from the distalfallopian tube (13). Type II cancers almost always harbor TP53 mutations(14). Also unlike type I cancers, which are relatively chemo-resistantand more often treated only with surgical excision, type II cancersrespond to conventional chemotherapy, particularly after maximaldebulking to reduce tumor burden (15, 16).

“Borderline” or “atypical proliferative” tumors lie in the middle ofthis spectrum, between the malignant cancers and the relatively harmless(non-neoplastic or benign) lesions. They are distinguished fromcarcinomas by the absence of stromal invasion and are precursors of typeI cancers. In light of their potential for malignancy, the standard ofcare for borderline tumors is surgical excision. Following surgery, theprognosis is excellent compared to ovarian cancers, with 5-year survivalrates over 85% (17). A minor but significant portion of borderlinetumors recur after surgery, however, and a subset of the recurrences arefound to have advanced to type I cancers (18). This progression isconsistent with molecular findings: serous borderline tumors typicallyexhibit mutations in BRAF or KRAS, like their malignant counterparts(low-grade serous carcinoma) (19, 20). The presence of a BRAF mutationin a borderline tumor is associated with better prognosis and a lowprobability of progression to carcinoma (21). In contrast, KRASmutations are associated with the progression to type I cancers (22).

The examination of fluids from pancreatic, renal, and thyroid cysts isroutinely used in clinical management (23-25). The fluids havehistorically been studied by cytology to identify malignant cysts.Ovarian cysts share many features with these other types of cysts, inthat they are common, often diagnosed incidentally, and are nearlyalways benign. However, aspiration of ovarian cyst fluid for cytology isnot standard-of-care. From a historical perspective, the difference indiagnostic management probably lies in the fact that cytology has notproven to be very informative for ovarian cysts, particularly fordistinguishing benign vs. borderline tumors (26, 27).

More recently, genetic analysis of specific types of cyst fluids hasbeen considered as an aid to cytology, given that conventional cytologyoften has limited sensitivity and specificity (28). Based on theemerging success of the molecular genetic evaluation of other types ofcysts, we reasoned that a similar approach could be applied to ovariancysts. Evaluation of DNA from cells and cell fragments shed into thecyst fluid would presumably allow the identification of tumor-specificmutations. Unlike other, conventional markers of neoplasia such asCA-125, cancer gene mutations are exquisitely specific indicators of aneoplastic lesion (29). Moreover, the type of mutation can in some casesindicate the type of neoplastic lesion present (30). Yamada et al. havedemonstrated that mutations can be detected in the cystic fluid ofovarian tumors by querying exons 4 to 9 of TP53, achieving sensitivitiesof 12.5% and 10%, for borderline and malignant tumors, respectively(31). Extremely sensitive methods for mutation detection, capable ofidentifying one mutant template allele among thousands of normaltemplates in a panel of genes, have recently been developed (32-34). Inthis study, we here applied one of these technologies to determinewhether mutations could be identified in ovarian cyst fluids, and if so,whether they provided information that could in principle be used indiagnosis and management.

Because there is currently no reliable way to determine whether anovarian cyst is malignant prior to surgical excision, many women undergounnecessary, invasive surgeries for non-malignant lesions. There is aneed in the art for techniques to determine whether surgery is requiredor unnecessary.

SUMMARY OF THE INVENTION

According to one aspect of the invention a method is provided in whichovarian cyst fluid is tested for mutations in a panel of genesfrequently mutated in ovarian neoplasms, wherein the panel comprisesBRAF, KRAS, and TP53.

According to another aspect of the invention a method is provided inwhich ovarian cyst fluid is tested for mutations in a panel of genesfrequently mutated in ovarian neoplasms, wherein the panel comprisesBRAF, KRAS, TP53, and one or more of AKT1, APC, BRCA1, BRCA2, CDKN2A,EGFR, FBXW7, FGFR2, MAPK1, NRAS, PIK3R1, and POLE.

According to another aspect of the invention a method is provided inwhich ovarian cyst fluid is tested for mutations in a panel of genesfrequently mutated in ovarian neoplasms, wherein the panel comprisesBRAF, KRAS, TP53, and one or more of CTNNB1, PIK3CA, PTEN, ARID1A, andPPP2R1A.

According to an additional aspect of the invention a method is providedin which ovarian cyst fluid is tested for mutations in a panel of genesfrequently mutated in ovarian neoplasms, wherein the panel comprisesBRAF, KRAS, TP53, AKT1, APC, BRCA1, BRCA2, CDKN2A, EGFR, FBXW7, FGFR2,MAPK1, NRAS, PIK3R1, and POLE.

According to an additional aspect of the invention a method is providedin which ovarian cyst fluid is tested for mutations in a panel of genesfrequently mutated in ovarian neoplasms, wherein the panel comprisesBRAF, KRAS, TP53, CTNNB1, PIK3CA, PTEN, ARID1A, and PPP2R1A.

According to an additional aspect of the invention a method is providedin which ovarian cyst fluid is tested for mutations in a panel of genesfrequently mutated in ovarian neoplasms, wherein the panel comprisesBRAF, KRAS, TP53, AKT1, APC, BRCA1, BRCA2, CDKN2A, EGFR, FBXW7, FGFR2,MAPK1, NRAS, PIK3R1, POLE, CTNNB1, PIK3CA, PTEN, ARID1A, and PPP2R1A.

These and other embodiments which will be apparent to those of skill inthe art upon reading the specification provide the art with powerfulmethods for assessing ovarian cysts without recourse to unnecessarysurgeries.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Schematic showing classes of ovarian cysts and the diagnosticpotential of the cyst fluid. Ovarian cysts and tumors are currentlyclassified according to microscopic evaluation after surgical removal.The majority of ovarian cysts are non-neoplastic (often “functional” inpremenopausal women). Ovarian tumors with combined cystic and solidcomponents are either benign tumors, borderline tumors, or malignantcancers (type I or II). Only cysts associated with borderline tumors andcancers require surgical excision. We show here that the DNA purifiedfrom cyst fluid can be analyzed for somatic mutations commonly found intheir associated tumors. The type of mutation detected not onlyindicates the type of tumor present but also could inform management.

FIGS. 2A-2B. Mutant allele fractions. (FIG. 2A) Classification by tumortype. No mutations were found in the DNA of non-neoplastic or benigncysts (red). Of the cysts that required surgery (blue), the medianmutant allele fraction was higher in the cyst fluids associated withtype II cancer (60.3%) than type I (7.8%) or borderline tumors (2.4%).(FIG. 2B) Classification by tumor stage. The DNA from cyst fluids oflate-stage cancers had a higher median mutant allele fraction (51.2%)than those of early-stage cancers (7.4%) or borderline tumors (2.4%).Horizontal bars depict median and IQR.

FIGS. 3A-3D. (Table 1.) Patient demographics. The clinicalcharacteristics of patients in this study and their tumorcharacteristics are indicated.

FIG. 4A-4E. (Table 2.) Mutations identified in tumors and cyst fluids.The mutations, mutant allele fractions, and amount of DNA recovered fromcyst fluids are indicated.

FIG. 5 (Table 3.) Detection of tumor-specific mutations in cyst fluid.The fraction of samples detected and the median fraction of mutantalleles are indicated, grouped by cyst type, cancer stage, and the needfor surgery.

FIG. 6 (Table 4.) Multivariate analysis for markers associated with needfor surgery. The presence of a mutation, cyst DNA amount, and commonserum biomarkers for ovarian cancer were analyzed for association withcysts that require surgical removal (Firth's penalized likelihoodlogistic regression).

FIG. 7A-7C (Fig. S1.) Markers associated with the need for surgery. CystDNA amount and levels of commonly used ovarian cancer serum biomarkersare plotted according to the cyst type and need for surgery. (FIG. 7A)The amounts of DNA in cyst fluids was generally higher in cystsrequiring surgery (blue) than those that do not (red), but nosignificant correlation was found (p=0.69). (FIG. 7B) CA-125 levels weresignificantly higher in cysts that required surgery than those that donot (p=0.01). (FIG. 7C) Serum HE4 levels was not correlated with theneed for surgery (p=0.92). P-values were calculated using Firth'spenalized likelihood logistic regression in a multivariate model (SeeExample 1).

FIGS. 8A-8H (Table S1) Primer sequences used in multiplex assay; Forwardprimers (SEQ ID NO: 1-133); Reverse primers (SEQ ID NO: 134-266).

FIG. 9A-9B Mutated genes found in the cyst fluid samples. FIG. 9A showsnon-neoplastic, benign, and borderline. FIG. 9B shows malignant Type Iand malignant Type II. Yellow boxes represent mutations with mutantallele frequency (MAF) between 0.1% and 1%; orange boxes representmutations with MAF between 1 and 10%; red boxes represent mutations withMAF greater than 10% (* indicates patients with insufficient DNA foranalysis; ** indicates patients with two detected mutations).

DETAILED DESCRIPTION OF THE INVENTION

The inventors have developed an assay for testing cyst fluids. Cystfluids are typically aspirated by a needle, preferably a fine needle.The aspiration can be performed under the guidance of a radiologicaltechnique, such as ultrasound. Other guidance techniques can be used asconvenient. Cyst fluids can typically be collected from any type ofovarian cyst or cystic neoplasm, and the term “cyst” is used here torefer to all types of ovarian growths with a cystic component.

Non-neoplastic ovarian cysts typically do not require surgical removaland do not display mutations. In contrast, ovarian cysts that areassociated with malignancy do require surgical removal and frequentlydisplay mutations; these mutations can further indicate the type andseverity of the disease. Testing for a panel that includes markers for abroad range of ovarian cysts permits the identification of cyst type andprognosis. It also permits a clinical decision to surgically remove ornot.

Other markers and clinical indication can be used in combination withthe ovarian cyst fluid assay results. Plasma markers such as CA-125 andHE4 can be assessed in patient plasma. Other protein or genetic markerscan be used in conjunction with the ovarian cyst fluid assay. Otherclinical indicators, including radiological findings and physicalfindings may be used in conjunction with the ovarian cyst fluid assay.

Testing may be performed using any technique that is targeted forparticular genes. These are not techniques that screen for any and allgene mutations. Rather, they are designed to detect mutations in certainpredetermined genes. In some cases they are designed to detect certainmutations or mutations in certain codons. Any analytic technique can beused for detecting mutations as is convenient, efficient, andsufficiently sensitive to detect mutations in ovarian cyst fluid. Theassays may be hybridization based, such as using specific probes orspecific primers. The assays may employ labeled probes or primers. Theassays may employ labeled secondary reagents that permit the primaryreagents to be detected. Such labels include radiolabels, fluorescentlabels, enzymatic labels, chromophores, and the like.

A variety of different mutation types can be detected and may be usefulin providing prognosis or management decisions. Such mutations includeLOH, point mutations, rearrangements, frameshifts, point mutations, andcopy number variations. Specific detection techniques for these mutationtypes or generic detection techniques may be used. It may be desirableto use control samples from other parts of the patient's body, such as abody fluid, like plasma, saliva, urine, feces, and the like.Alternatively other control samples may include tissues such as normaltissue from a non-ovary, or cells or tissues from the ovarian cyst wall.

Cyst fluid may be obtained by any technique known in the art, includingbut not limited to needle aspiration. The aspiration may optionally beguided by a radiological technique such as ultrasound. Cyst fluid may beaspirated before or after initial surgical removal or subsequentsurgical removal.

In some embodiments, primers will incorporate unique identification DNAsequence (UID) which are molecular barcodes. These can be randomlygenerated and attached to templates as a means to reduce errors arisingfrom amplification and sequencing. Probes, primers, and UIDs canincorporate non-naturally occurring modifications to DNA sequences, byinternucleotide linkage modifications, by sugar modifications, and bynucleobase modifications. For example, phosphorothioate (PS) linkagescan be used in which sulfur substitutes for one nonbridging phosphateoxygen. This imparts resistance to nuclease degradation. Othermodifications which can be used include N3′ phosphoramidate (NP)linkages, Boranophosphate internucleotide linkages, Phosphonoacetate(PACE) linkages, Morpholino phosphoramidates, Peptide nucleic acid(PNA), 2′-O-Me nucleoside analog, 2′F-RNA modification,2′-deoxy-2′-fluoro-β-D-arabino nucleic acid (2′F-ANA) modification andLocked nucleic acid (LNA).

Other techniques which are unbiased toward particular genes can be usedas well for assessing genes of interest in cyst fluid. Such techniquesinclude whole-genome or whole exome techniques. These may includeassessments by nucleotide sequencing. The nucleotide sequencing may beredundant nucleotide sequencing. Targeted sequencing methods can be usedas well.

The methods described here achieve high degrees of sensitivity andspecificity. Sensitivity may be at least 15%, at least 20%, at least25%, at least 50%, at least 60%, at least 70%, at least 80%, at least85%, at least 90%, at least 95% for borderline and malignant tumors.Specificity may be at least 15%, at least 20%, at least 25%, at least50%, at least 60%, at least 70%, at least 80%, at least 85%, at least90%, at least 95% for borderline and malignant tumors.

Removal of ovarian cyst fluid assay from the body can be accomplishedbefore any surgery occurs. Thus the results of the assay can help guidethe decision to perform surgery. If surgery has occurred to remove theovarian cyst, and if it returns, a sample of ovarian cyst fluid may beobtained from the body at that time. The assays will typically beperformed in a clinical laboratory on samples that have been removed bya skilled clinician, such as an interventional radiologist or a surgeon.The samples may be assayed immediately or they may suitable stored andor shipped for testing. It is possible that DNA will be extracted fromthe sample prior to shipping it to a laboratory for testing. Resultswill generally be communicated back from the assaying laboratory to theclinician for communication to a patient. Results may be recorded inpaper or electronic medical records.

Ovarian cancer is the most lethal gynecologic cancer in women. Howeverscreening is not recommended by the U.S. Preventive Services Task Forceusing current diagnostic approaches, which too frequently lead to“important harms, including major surgical interventions in women who donot have cancer” (Moyer and Force, 2012). We have demonstrated here thatdriver mutations in ovarian tumors are also present in their associatedcyst fluids. Moreover, the mutant allele frequencies in the cyst fluidsare relatively high (median 12.6%, IQR of 2.7% to 40.2%), facilitatingtheir detection. There were no mutations detected in the cyst fluidsthat were not also present in the tumors, and vice versa. Alsoimportantly, no mutation was identified in non-neoplastic cysts or cystsassociated with benign tumors. Overall, mutations were detected in amajor fraction (87%) of cysts requiring surgery but not in any cyst thatdid not require surgery.

Our results demonstrate that mutations present in ovarian tumors arealso present in their associated cyst fluids. Moreover, the mutantallele frequencies in the cyst fluids are relatively high (median 12.6%,IQR of 2.7% to 40.2%), facilitating their detection. There were nomutations detected in the cyst fluids that were not also present in thetumors, and vice versa. And most importantly, mutations were detected ina major fraction (85%) of cysts requiring surgery but not in any cystthat did not require surgery (Tables 2 and 3).

Although most (85%) of the 55 cysts requiring surgery had detectablemutations in their fluidic compartment, eight did not. All of theseeight cysts occurred in borderline tumors or type I cancers, whilemutations were always (100%) detectable in type II cancers (Tables 2 and3). There are two potential explanations for our failure to detectmutations in these eight cysts. First, it is possible that the mutantDNA concentration in these cysts was below the level of technicalsensitivity of our assay (˜0.1% mutant allele fraction). We excludedthis possibility by evaluating the tumors themselves: no mutations weredetected in any of the tumors from these 8 patients. The second, andtherefore more likely explanation, is that our panel of 133 amplicons,containing regions of 17 genes, was not adequate to capture themutations that were present. Unlike type II cancers, which nearly alwayscontain TP53 mutations (94% of the type II cancers we studied, forexample), the genomic landscapes of type I cancers and borderline tumorsare more heterogeneous and not as well studied (II). Further geneticevaluation of these tumors should facilitate the incorporation ofadditional amplicons in the panel to reach higher sensitivities.Nevertheless, the 100% sensitivity for type II cancers in our study ishighly encouraging, given that these cancers account for over 90% ofovarian cancer deaths.

One limitation of our study is the number of patients evaluated. Thoughexcision of ovarian cysts is one of the most commonly performed surgicalprocedures, banking of cyst fluids is not common, even in academiccenters. Thus, we only had relatively small numbers (n=22) ofnon-neoplastic cysts and benign tumors available for study. Even so, thedifferences in genetic alterations among the various cyst types werestriking (Tables 2 and 3). Our study will hopefully stimulate collectionand analyses of ovarian cyst fluids that will be able to establishsmaller confidence limits around the sensitivities and specificitiesreported in the current study.

A potential clinical limitation of our approach is the concern bygynecologists that needle puncture of a malignant ovarian cyst leads toseeding of the peritoneum. This concern is based on inconclusiveevidence about the dangers of cyst rupture during surgery and is, atbest, controversial (40). Moreover, leakage is expected to be much lesslikely when a tiny needle is inserted into the cyst underultrasound-guidance than when cysts are manipulated during surgery. Theidea that malignant cysts might shed cancer cells if needle-puncturedalso seems incongruent with the widespread practice of laparoscopicremoval of ovarian cysts (41). Laparoscopic removal of a cyst carries arisk of cyst rupture, perhaps higher than needling (42). Finally,malignant pancreatic cysts are at least as dangerous as malignantovarian cysts, yet the standard-of-care for pancreatic cysts involvesrepeated sampling of cyst fluid through endoscopic ultrasound over manyyears (43, 44). Though pancreatic cysts and ovarian cysts lie indifferent anatomical compartments, it is encouraging that aspiration ofpancreatic cysts is not associated with an increased risk of mortalityin patients with pancreatic cancer (45). Finally, recent advancements inmethods to plug biopsy tracts, using materials such as absorbablegelatin slurry and torpedo, can further decrease the risk of tumorspillage associated with fine-needle aspirations (46, 47). On the basisof these observations and recent developments, we believe thatultrasound-guided aspiration of ovarian cyst fluids would likely be asafe and well-tolerated procedure.

As noted in the background of the invention section above, seven to tenpatients with benign ovarian cyst lesions undergo surgery for each caseof ovarian cancer found (48). In addition to the psychological impact apotential diagnosis of cancer has on patients, surgery for benignlesions entails considerable cost and morbidity. OVA-1 is the onlyFDA-cleared test to date that aims to distinguish benign versusmalignant adnexal mass. It measures levels of five serum markers(CA-125, (3-2 microglobulin, apolipoprotein A1, prealbumin, andtransferrin) and is used to stratify patients who should consult agynecologic oncologist rather than a general gynecologist for surgery.However the test has a specificity of 43% for ovarian cancer, which iseven lower than that of CA-125 alone (49). While the test mightencourage patients with suspected ovarian cancer to seek specializedcare, it would not decrease the number of unnecessary surgeries forwomen with benign adnexal masses.

This study was driven by the need for a biomarker that would helpdistinguish malignant ovarian tumors from benign lesions and therebyreduce the number of unnecessary surgeries. Such distinction is oftendifficult based on symptoms and conventional diagnostic criteria. Forexample, in a large study of 48,053 asymptomatic postmenopausal womenwho underwent ultrasound examination by skilled sonographers, 8 (17%) ofthe 47 ovarian cancers that were identified occurred in women withpersistently normal sonographic findings (Sharma et al., 2012). Alleight cases were type II cancers, highlighting the potential utility ofan additional assay to detect this highly lethal and aggressive type ofovarian cancer. On the other hand, of the 4367 women with abnormal sonographic findings, less than 1% of cases proved to have malignancy uponsurgery. Furthermore, of the 32 women with borderline or Type I cancersdiagnosed, 22 (69%) had a serum CA-125 level within the clinicallyaccepted normal range (≤35 units/mL). In our study, 18 of 18 (100%) typeII cancers were detectable by virtue of the mutations found in cystfluid DNA while none of the 18 benign or non-neoplastic cyst fluidcontained such mutations. It is also important to note that the readoutof our assay is quantitative and not dependent on the skill level of thereader (in contrast to sonography). Finally, the procedure can beperformed minimally invasively in an outpatient setting. The goal of ourtest is not to replace clinical, radiologic, or sonographic evaluationbut to augment them with molecular genetic markers.

Our study, though only proof-of-principle, illustrates one route toimprove management of patients with ovarian cysts. Genetic analysis isnot the only such route; proteomics could also provide clues to thecorrect diagnosis (50, 51). One can easily imagine how such additionalinformation could be used to inform clinical practice in conjunctionwith current diagnostic methods. For example, if a cyst contained lowamounts of DNA, no detectable mutations, and if the patient had lowCA-125 levels, our data suggest that it is very unlikely to be aborderline tumor or malignant lesion. Either no surgery, or laparoscopicrather than open surgery, could be recommended for that patient, even ifthere was some solid component upon imaging. The option to avoid surgerywould be particularly valuable for pre-menopausal women who generallyhave a low risk of ovarian cancer and might wish to preserve theirfertility, as well as patients who are poor surgical candidates.However, our assay in its current format cannot completely rule outmalignancy because a fraction of early-stage cancer patients did nothave detectable mutations in their cysts. Therefore, patients whoseclinical and functional status allows them to undergo surgery andanesthesia might still choose to have a surgical procedure. On the otherhand, a minimally invasive test that provides additional, orthogonalinformation to patients and surgeons could inform their decision aboutthe advisability of surgery.

Our data suggest that a cyst without any solid component upon imaging,and thereby unlikely via conventional criteria to be malignant, shouldbe removed promptly if the cyst fluid contained a TP53 mutation.Radical, rather than conservative, surgery might be appropriate due tothe high likelihood of an aggressive type II cancer. In contrast, if aBRAF mutation was identified, the lesion is presumably a borderline orlow-grade tumor; thus conservative rather than radical surgery might besufficient. Lastly, given that certain types of ovarian cancers (typeII) tend to respond well to chemotherapy while others (type I) arerelatively chemo-resistant, knowing the type of cancer present prior tosurgery based on the mutation profile could help guide decisionsregarding the use of neoadjuvant chemotherapy. Validation of these datain a much larger, prospective trial will of course be required beforeincorporation of this approach into clinical practice.

The above disclosure generally describes the present invention. Allreferences disclosed herein are expressly incorporated by reference. Amore complete understanding can be obtained by reference to thefollowing specific examples which are provided herein for purposes ofillustration only, and are not intended to limit the scope of theinvention.

EXAMPLE 1 Materials and Methods Patient Samples

Cyst fluids were collected prospectively from 77 women presenting with asuspected ovarian tumor. Patients were diagnosed by transvaginalsonography or computed tomography and admitted for surgical removal ofthe cyst by gynecologic oncology surgeons at Sahlgrenska UniversityHospital, Gothenburg, Sweden. The study was approved by the ethicalboard of Gothenburg University and patients provided written consent.According to the approved protocol, ovarian cyst fluids were collectedafter removal of the cyst from the abdomen. All samples were immediatelyput in 4° C. for 15-30 minutes, centrifuged for 10 minutes at 500 g, andaliquoted into Eppendorf tubes. The fluids were transferred to −80° C.,within 30-60 minutes after collection. All histology was reviewed byboard-certified pathologists (Table 1).

Plasma HE4 concentrations were determined using a commercial HE4 EIAassay (Fujirebio Diagnostics) and plasma CA-125 levels were measuredusing the Architect CA 125 II (Abbott Diagnostics, USA). DNA waspurified from tumor tissue (either freshly-frozen, or formalin-fixed andparaffin-embedded) after microdissection to remove neoplasticcomponents. DNA was purified from tumors and from cyst fluids using anAllPrep DNA kit (Qiagen) according to the manufacturer's instructions.Purified DNA from all samples was quantified as previously described(52).

Statistical Analysis

A Wilcoxon rank-sum test was used to compare the amount of DNA in thecancers and borderline tumors with the amount of DNA in the simple cystsand benign tumors. The fraction of samples detected by tumor-specificmutations in the cyst fluid, as well as their 95% confidence intervals,was calculated for each tumor type (Table 3). When the presence of amutation in the cyst fluid was used to predict the need for surgery, thesensitivity and specificity of the test, as well as their 95% confidenceintervals, were calculated. Firth's penalized likelihood logisticregression was used to quantify the association between molecularfeatures of cyst fluids and the need for surgery (Table 4) in amultivariate model. The model predictors included the presence ofmutation, log10 (ng) of cyst DNA and indicators for normal CA-125 andHE4 values. Normal CA-125 values were defined as <35 U/mL and normal HE4values were defined as <92 pmol/L and <121 pmol/L for pre- andpost-menopausal women, respectively. Statistical analyses were performedusing the R statistical package (version 3.1.2). Unless noted otherwise,all patient-related values are reported as means±SD.

Mutation Detection and Analysis

DNA from either cyst fluids or tumors was used for multiplex PCR, aspreviously described (34). One-hundred-and-thirty-three primer pairswere designed to amplify 110 to 142 bp segments containing regions ofinterest from the following 17 genes: AKT1, APC, BRAF, CDKN2A, CTNNB1,EGFR, FBXW7, FGFR2, KRAS, MAPK1, NRAS, PIK3CA, PIK3R1, POLE, PPP2R1A,PTEN, and TP53. Primer sequences are listed in Table S1. These primerswere used to amplify DNA in 25 μL reactions as previously described(34). For each sample, three multiplex reactions, each containingnon-overlapping amplicons, were performed. Reactions were purified withAMPure XP beads (Beckman Coulter) and eluted in 100 μL of Buffer EB(Qiagen). A fraction (2.5 μL) of purified PCR products were thenamplified in a second round of PCR, as described (34). The PCR productswere purified with AMPure and sequenced on an Illumina MiSeq instrument.

We used Safe-SeqS, an error-reduction technology for detection of lowfrequency mutations as described to distinguish better between genuinemutations in the samples and artifactual variants arising fromsequencing and sample preparation steps, (34). High quality sequencereads were selected based on quality scores, which were generated by thesequencing instrument to indicate the probability a base was called inerror. The template-specific portion of the reads was matched toreference sequences. Reads from a common template molecule were thengrouped based on the unique identifier sequences (UIDs) that wereincorporated as molecular barcodes. Artifactual mutations introducedduring the sample preparation or sequencing steps were reduced byrequiring a mutation to be present in >90% of reads in each UID family(i.e., to be scored as a “supermutant”). In addition, DNA from normalindividuals was used as a control to identify potential false positivemutations (see main text). Only supermutants in samples with frequenciesfar exceeding their frequencies in control DNA samples (i.e., >mean+5standard deviations) were scored as positive.

EXAMPLE 2 Characteristics of the Tumors and Cyst Fluid Samples

DNA was isolated from surgically excised ovarian cysts of 77 women. Tenof them had non-neoplastic cysts, 12 had benign tumors, 24 hadborderline tumors, and 31 had cancers (13 Type I and 18 Type II). Age,histopathologic diagnosis, stage, and other clinical information areprovided in Table 1. The median amount of DNA recovered from the cystswas 222 ng (interquartile range (IQR) of 53 to 3120 ng) (Table 2). Therewas no significant difference in the amounts of DNA between borderlinetumors and type I or type II cancers (Table 2). However, the borderlinetumors and cancers contained significantly more DNA than thenon-neoplastic cysts or benign tumors (4453±6428 ng vs. 62±64 ng;p<0.001, Wilcoxon rank-sum test).

EXAMPLE 3 A Multiplex PCR-Based Test to Identify Tumor-SpecificMutations in Cyst Fluid Samples

We designed a multiplex PCR-based test that could simultaneously assessthe regions of 17 genes frequently mutated in ovarian tumors. The amountof DNA shed from neoplastic cells was expected to be a minor fraction ofthe total DNA in the cyst fluid, with most DNA emanating from normalcells. We therefore used a sensitive detection method, called Safe-SeqS(Safe-Sequencing System), to identify mutations in cyst fluid samples(34). In brief, primers were designed to amplify 133 regions, covering9054 distinct nucleotide positions within the 17 genes of interest(Table S1). Three multiplex PCR reactions, each containingnon-overlapping amplicons, were then performed on each sample. Oneprimer in each pair included a unique identifier (UID) for each templatemolecule, thereby drastically minimizing the error rates associated withPCR and sequencing, as described previously (34) (Table S1). Under theconditions used in the current experiments, mutations present in >0.1%of template molecules could generally be reliably determined. We couldnot perform sequencing on five cysts (two simple cysts, twocystadenomas, one borderline tumor) because there was insufficient DNA(<3 ng recovered), and these were scored in a conservative fashion, as“negative” for mutations. When this test was applied to the 22 cystfluids obtained from patients with simple cysts (n=9) or benign tumors(n=13), no mutations were identified (Tables 2 and 3). This was in starkcontrast to the fluids obtained from the 18 patients with type IIcancers, all of which were found to contain a mutation (Tables 2 and 3).Ten (77%) of the 13 cyst fluids from patients with type I cancers and 19(79%) of the 24 cyst fluids from patients with borderline tumorscontained at least one detectable mutation. When categorized by the needfor surgery (i.e., presence of a borderline tumor or a type I or type IIcancer), the sensitivity of this test was 85% (47 of 55 cysts; 95%confidence interval of 73% to 94%) and the specificity was 100% (95%confidence interval of 78% to 100%; Table 3).

Ovarian cancers are generally detected only late in the course ofdisease, explaining the poor prognosis of patients. Accordingly, only 11of the 31 cysts associated with cancers in our study had early (Stage Ior II) disease (Table 1). As expected, most of these were type Icarcinomas (n=8). Nevertheless, it was encouraging that mutant DNA couldbe detected in nine (82%) of these 11 patients (Table 3). Mutationscould be detected in 95% of the 20 patients with Stage III or IV cancers(Table 3).

A variety of control experiments were performed to confirm the integrityof these results. One informative positive control was provided by theresults of sequencing of DNA from the tumors, using the identical methodused to analyze DNA from the cyst fluids. Fifty-three of the 55borderline and malignant cases had tumor available for this purpose.Every mutation identified in a tumor was found in its cyst fluid, andvice versa. As expected, the mutant allele frequencies in the tumorswere often, but not always, higher than in the cyst fluid (Table 2). Asanother positive control, we used an independent PCR and sequencingreaction to confirm each of the cyst fluid mutations listed in Table 2.This validated not only the presence of a mutation, but also confirmedits fractional representation. The median relative difference betweenthe fractions of mutant alleles in replicate experiments was 7.0% (IQRof 3.5% to 8.9%). Finally, four patients were found to have twoindependent mutations (Table 2). For example, the cyst fluid of patientOVCYST 081, who had high-grade endometrioid carcinoma, had a missensemutation (R280K) in TP53 plus an in-frame deletion of PIK3R1 at codons458 and 459 of PIK3R1. The TP53 mutation was found in 3.0% of alleleswhile the PIK3R1 mutation was found in 3.7% of the alleles analyzed.Similar mutant allele frequencies among completely different mutationsin the cyst fluid of three other patients provided further indicators ofreproducibility (Table 2). All genetic assays were performed in ablinded manner, with the operator unaware of the diagnoses of thepatients from whom the cyst fluids were obtained.

In addition to DNA from normal individuals used as controls, additionalnegative controls were provided by the simple cysts and benign tumors.Using the identical assay, none of the DNA from their cyst fluidscontained detectable mutations (Table 2). A final control was providedby the borderline and malignant tumors themselves. In general, only oneor two of the 9054 base-pairs (bp) queried were mutated in any one tumor(Table 2). The other 9000 bp could then be independently queried in thecorresponding cyst fluid, and none of these positions were found to bemutated.

EXAMPLE 4 Relationship Between the Type of Tumor Present and the Type ofMutation Found in the Associated Cyst Fluid Sample

The mutant allele fractions in the cyst fluids tended to be higher inthe type II cancers (median of 60.3%) than the type I cancers (median of7.8%) or borderline tumors (median of 2.4%), though there wasconsiderable overlap (Tables 2 and 3). On the other hand, the type ofmutation varied considerably among these cysts. In type I tumors, thegenes mutated were BRAF (n=1), KRAS (n=5), NRAS (n=1), PIK3R1 (n=1),PPP2R1A (n=1), PTEN (n=1), or TP53 (n=3). Two distinct mutations werefound per sample in three type I cancers. One type I cancer had a BRAFmutation. This BRAF mutation (V600_S605>D) is unusual that it resultedfrom an in-frame deletion/insertion rather than the base substitution(V600E) characteristic of the vast majority of BRAF mutations reportedin the literature. This mutation has been observed in a papillarythyroid cancer and a cutaneous melanoma (35, 36). The deletion resultsin loss of a phosphorylation site in the activation loop of BRAF, whilethe insertion of an aspartic acid has been suggested to increase BRAFkinase activity by mimicking an activating phosphorylation (37). Incontrast, all but one type II cancers (94% of 18) had mutations in TP53;the only exception was OVCYST 081, a high-grade endometrioid carcinoma.The borderline tumors were distinguished by yet a different pattern fromthat of the either type I or type II cancers. Of the 19 mutations inborderline tumors, 12 (63%) were at BRAF V600E, never observed in type Ior type II cancers, and the remainder were at KRAS 12 or 61 (Table 2).

EXAMPLE 5 Markers Associated with the Need for Surgery

A multivariate analysis was used to identify the most informativemolecular features of cyst fluids and to compare them to the commonlyused serum biomarkers for ovarian cancer, HE4 (human epididymis protein4) and CA-125 (38, 39) (Table 4). We defined “informative” as indicatinga need for surgery (i.e., borderline tumors or type I or II cancers).The amount of DNA in cyst fluids was generally, but not significantly,higher in the cysts requiring surgery (p=0.69, Table 4), though therewere many cysts not requiring surgery that had higher DNA levels thancysts requiring surgery (Figure S1A). Similarly, the serum CA-125 levelswere significantly higher in cysts requiring surgery (p=0.01, Table 4),but there were many cysts not requiring surgery that had higher levelsthan those requiring surgery (Figure S1B). Serum HE4 levels were notcorrelated with cyst type (P=0.92, Table 4; Figure S1C). On the otherhand, the presence of a mutation was highly informative for the presenceof a cyst requiring surgery in the multivariate analysis, as nomutations were found in cysts not requiring surgery (P<0.001, Table 4).

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The disclosure of each reference cited is expressly incorporated herein.

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1-34. (canceled)
 35. A method, comprising: testing ovarian cyst fluidfor a mutation in at least one gene mutated in a type II ovarianneoplasm, wherein said at least one gene comprises TP53, wherein thestep of testing employs a step of adding a unique identifier (UID) to aplurality of TP53 template DNA molecules in the ovarian cyst fluid, andwherein the method has a sensitivity level of at least 70%.
 36. Themethod of claim 35, wherein said at least one gene further comprisesKRAS.
 37. The method of claim 35, wherein the step of testing employsTP53-specific reagents.
 38. The method of claim 35, wherein the step oftesting employs TP53 mutation-specific reagents.
 39. The method of claim35, wherein the testing is performed on the ovarian cyst fluid and on asample selected from the group consisting of cyst wall and normal,non-ovarian tissue.
 40. The method of claim 35, wherein the ovarian cystfluid is obtained by needle aspiration of an ovarian cyst.
 41. Themethod of claim 35, wherein the ovarian cyst fluid is obtained prior toany surgical removal of the ovarian cyst.
 42. The method of claim 35,wherein the ovarian cyst fluid is obtained after surgical removal of theovarian cyst and recurrence of the ovarian cyst.
 43. The method of claim35, wherein a copy number variation, a loss of heterozygosity, or both,is determined for said at least one gene.
 44. The method of claim 35,wherein a point mutation, a rearrangement, a frameshift, or combinationsthereof, is determined for said at least one gene.
 45. The method ofclaim 35, wherein the method has a sensitivity level of at least 85%.46. The method of claim 35, wherein the method has a specificity levelof at least 90%.
 47. The method of claim 35, wherein the ovarianneoplasm needs surgery.